Mannose-containing glycolipids which are sensitive to phosphatidyl-inositol (PI) specific phospholipases are found in eukaryotic cells both as free mannolipids (GPI-lipids) and as anchor moieties of selected membrane proteins (GPI-anchored proteins). Recent research from these and other laboratories has strongly indicated that certain of the free GPI-lipids are precursors of the GPI anchors and that lesions which interrupt their synthesis may account for the lack of expression of GPI-anchored proteins on murine Thy-1 negative T lymphoma mutants and on circulating red and white cells in the hemolytic anemia, Paroxysmal Nocturnal Hemoglobinuria (PNH). The proposed biochemical, cell and molecular biological studies will examine the structures and biosynthesis of both free and protein-associated GPI-lipids, vesicular transport of GPI-lipids and the possible role(s) of GPI-lipids an enzyme substrates and as sources of second messengers. In order to further elucidate the path of GPI-lipid biosynthesis, studies will focus especially on available and new human and murine cell mutants which do not express GPI-anchored proteins, including cells of patients suffering from Zellweger syndrome, which is characterized by deficient synthesis of ether lipids. Related studies will use stable reincorporation of GPI- anchored proteins into the plasma membrane of wild type cells to initiate a line of "cell therapy", add GPI-lipids onto the surface of mutant cells in an attempt to restore their ability to express GPI-anchored proteins, and clone genes which account for the lesions in GPI-lipid synthesis which are responsible for the lack of cell surface expression of such proteins in PNH.